Flow cytometry and immunofluorescence analyses of iPSC-derived neurons.

<p>A) Flow cytometry analysis of neuronal surface markers CD56 and CD24 at day 32 of differentiation. B) Representative phase contrast microscope image of cultured neurons at day 32 of differentiation. Scale bar: 100 µm. C) At day 45 of differentiation, cells express MAP2 (green) along soma and dendrites and the postsynaptic protein PSD95 (red), confirming the advanced stage of differentiation into neurons. Scale bar: 100 µm. D) Cells also express the forebrain-specific marker BF1 (green) and Tuj1 (red) at 45 days of culture. They are often co-localized (yellow). Scale bar: 50 µm.</p

Spontaneous calcium transients increase in iPSC-derived neurons during development.

<p>A) After imaging of spontaneous Ca²⁺- transients iPSC-derived neurons were fixed and stained for Tuj1 at day 32 and day 55, revealing more complex morphology at day 55 (scale bar = 40 µm). B) Representative Fluo-4 spontaneous Ca²⁺- transients from 3 iPSC-derived neurons at day 32 and at day 55 (ΔFluo = ΔFluorescence; a.u. = arbitrary unit). Neurons exhibit slow broad transients, consistent with early developing neurons at day 32. By day 55, calcium transients take on a more spike-like morphology reflecting what has been reported for maturing neurons. Group data demonstrates that iPSC-derived neurons exhibit a significant increase in calcium transients at day 55 as compared to day 32 (n = 113 neurons/10 coverslips for both groups, p<0.001 comparing day 32 with day 55). C) The average number of Ca²⁺ transients at day 55 was dramatically reduced following perfusion with 1 µM TTX with recovery following washout of the toxin (n = 20/3 coverslips, p<0.001 compared with average number of Ca²⁺ transients prior to TTX perfusion). A sample trace derived from a representative experiment is shown on the left).</p

iPSCs-derived neurons plated with glia express different electrophysiological properties than neurons plated on POL.

<p>A) Representative APs in response to step current injections of 20 pA in current clamp mode for a cell at day 55 co-cultured with mouse glia. APs were observed on 13 out of 15 cells, and in 4 out of the 13 cells with APs rebound APs at the end of hyperpolarizing current injections were also present. B) Examples of mEPSCs recorded in voltage clamp configuration. Cells were plated on mouse glia by mixing them before plating. They were held at −70 mV. TTX at a concentration of 1 µM was added to the bath solution to suppress spontaneous excitation and to allow isolation of synaptic events induced by spontaneous transmitter release.</p

Evolution of active membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) Representative traces of voltage clamp recordings showing fast inward currents followed by long-lasting outward currents in a cell at day 55, due to voltage steps in 10 mV increments (shown in the upper panel). The inset shows a high magnification view of the inward current. Inward Na⁺ currents were observed in 13 out of the 22 cells. Following initial recording, cells were perfused with 1 µM TTX to block Na⁺ currents, and subsequently with 10 mM TEA (tetraethylammonium) to block K⁺ currents. B) The percentage of cells showing APs in response to step current injections of 20 pA over the total number of cells recorded in the same day. C) The graph shows the increase over time of the average amplitude of the inward Na⁺ current measured at the peak (pA). D) The graph shows a decrease over time of the Full Width at Half Maximum (FWHM) (ms) of APs. E) The graph shows an increase of the AP amplitude (mV) over time. F) Evolution of the AP threshold (mV) over time. G) Evolution of the average peak K⁺ currents over time (pA).</p

Electrophysiological parameters in neurons plated on POL and mouse glia co-cultures at day 55.

<p>The table summarizes the differences between various electrophysiological properties in neurons plated on POL (n = 20) and mouse-glia co-cultures (n = 15) at day 55. Values are expressed as mean and SE (standard error).</p

Evolution of basal membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) The average resting membrane potential (RMP in mV) decreased over time. The number of cells examined for these experiments are shown inside the bar in these and the following figures. The vertical lines correspond to the SEM in this and the following figures. **: p<0.001; *: 0.0010.05 for these and the following figures. B) The membrane input resistance decreased over time (GΩ). C) The membrane time constant decreased over time (ms).</p

Reduced Susceptibility of DNA Methyltransferase 1 Hypomorphic (Dnmt1^N/+) Mice to Hepatic Steatosis upon Feeding Liquid Alcohol Diet

<div><h3>Background</h3><p>Methylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated.</p> <h3>Methodology/Principal Findings</h3><p>Dnmt1 wild type (<em>Dnmt1</em>^+/+) and hypomorphic (<em>Dnmt1</em>^N/+) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in <em>Dnmt1</em>^+/+ mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, <em>Dnmt1</em>^N/+ mice were less susceptible to alcoholic steatosis compared to <em>Dnmt1</em>^+/+ mice. Expression of several key genes involved in alcohol (<em>Aldh3b1</em>), lipid (<em>Ppara, Lepr, Vldlr, Agpat9</em>) and xenobiotic (<em>Cyp39a1</em>) metabolism, and oxidative stress (<em>Mt-1, Fmo3</em>) were significantly (P<0.05) altered in <em>Dnmt1</em>^N/+ mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of <em>Agpat9, Lepr, Mt1 and Ppara</em> were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice.</p> <h3>Conclusions/Significance</h3><p>Although feeding alcohol diet reduced Dnmtase activity, the loss of one copy of <em>Dnmt1</em> protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.</p> </div

miR-148 and miR-152, which target Dnmt1 and Dnmt3b, are upregulated in the livers of the wild type mice fed the liquid alcohol diet.

<p><b>A.</b> miR-148/152 cognate site predicted by TargetScan in the 3′-UTR of Dnmt1. <b>B.</b> Northern blot analysis demonstrated increased expression of miR-148 in mice fed alcohol. Total RNA (5 µg) was separated in 15% acrylamide-8 M urea gel, transferred to nylon membrane and subjected to Northern blotting using ³²P-labeted anti-miR-148 oligo as probe, washed and subjected to autoradiography. The blot was rehybridized to 5S rRNA probe after stripping to demonstrate comparable RNA in each lane. <b>C.</b> qRT-PCR analysis confirmed upregulation of hepatic miR-148&152 in mice fed alcohol. miR-148/152 and RNU6B were measured in the liver cDNAs using Taqman probes and primers specific for each miRs and the data was normalized to RNU6B level. <b>D.</b> Dnmt1 is a validated target of miR-148&152. Hepa cells were transfected with psiCHECK2 vector harboring wild type or mutant Dnmt1 3′-UTR downstream of renilla luciferase coding region along with 50 nM miR-148b/152 mimic or scrambled RNA (NC RNA). After 48 h, renilla (RLU2) and firefly luciferase (RLU1) (expressed from the same vector) activities were measured and the data are represented as RLU2/RLU1. Each assay was performed in triplicate. <b>E.</b> Upregulation of miR-148/152 in Hepa cells transfected with the respective miRs compared to the controls (NC RNA transfected cells). Total RNAs from each sample in D was subjected to qRT-PCR as described in B. Each assay was performed in triplicate. <b>F.</b> Endogenous DNMT1 and DNMT3b protein levels were reduced in cells expressing ectopic miR-148/152. H293T cells were transfected with 50 nM miRs or NC RNA. After 24 h, cells were split and harvested at indicated time points post-transfection for western blot analysis of whole cell extracts (100 µg) with specific antibodies. Relative expression was determined after normalization of the signal to that of Gapdh. Single, double and triple asterisks represent P-values ≤0.05, ≤0.01 and ≤0.005, respectively.</p